Publication Date
4-3-2023
Journal
Investigative Ophthalmology & Visual Science
DOI
10.1167/iovs.64.4.8
PMID
37036417
PMCID
PMC10103726
PubMedCentral® Posted Date
4-10-2023
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Mice, Animals, Epithelium, Corneal, Interleukin-18, Inflammasomes, NLR Family, Pyrin Domain-Containing 3 Protein, NF-kappa B, Mice, Inbred C57BL, Dry Eye Syndromes, Interleukin-1beta, dry eye; corneal epithelium, NF-kB; inflammasome; IL-1β; IL-18
Abstract
PURPOSE: To determine whether 24-hour exposure to the desiccating stress (DS) dry eye model induces NF-kB and NLRP3 inflammasome pathways in the mouse cornea epithelium.
METHODS: Six- to 8-week-old C57BL/6J mice were housed under normal humidity (nonstressed) or subjected to DS from a drafty, low-humidity environment combined with subcutaneous scopolamine four times/day for one day to suppress tear production (DS1). Cornea whole mounts were prepared for immunofluorescent staining, or the corneal epithelium was scraped for NF-kB p-p65 ELISA, Western blot, or real-time PCR to detect NF-kB and inflammasome pathway proteins and gene transcripts, respectively.
RESULTS: NF-kB phospho-p65 protein, nuclear NF-kB p-p65, and expression of the NF-kB inducible cytokines (IL-12a, IL-12b, and lymphotoxin b [Ltb]) and chemokine (CCL-2) genes were significantly increased in DS1 compared to nonstressed control. NLRP3 protein and RNA transcripts significantly increased in DS1. NLRP3 and Caspase-1 immunostaining increased in the cornea epithelium at DS1. At DS1 there was no change in IL-18 and a decrease in IL-1β mRNA transcripts; however, levels of bound and total IL-18 protein increased at DS1, and the level of mature IL-1β increased from DS1 to DS5.
CONCLUSIONS: These findings indicate innate NF-kB and NLRP3 inflammasome inflammatory pathways are induced in the corneal epithelium within one day in the DS dry eye model. NF-kB activation was associated with increased expression of inflammatory mediators involved in dry eye. Induction of these pathways is accompanied by increased bound/total IL-18 and mature IL-1β.